This project is aimed towards understanding the mechanisms which mediate and regulate Ca-2+ signaling in salivary gland cells. Neurotransmitter stimulation of fluid secretion in salivary glands is mediated via a biphasic elevation in cytosolic [Ca-2+]; an initial transient increase due to internal release and a latter sustained increase due to Ca-2+ influx. Sustained fluid secretion is directly dependent upon the sustained elevation of [Ca-2+] and thus on Ca-2+ influx. Recently, our efforts have been focused on the Ca-2+ influx mechanism in salivary gland cells, which appears to be a mediated via store-operated Ca-2+ entry (SOCE) that is ubiquitously present in many other non-excitable cells. The molecular mechanism(s) of this influx has not yet been determined in any cell type. Recently, the transient receptor potential (TRPC) family of ion channel proteins have been proposed as molecular components of the store-operated Ca-2+ influx channel (SOCC). However, the physiological function(s) of the presently identified TRPCs has not yet been fully established. By expressing TRPC1 in vivo in rat SMG by using an adenovirus encoding hTrp1 (AdHA-hTrp1) and in salivary gland cell lines, we had previously reported that TRPC1 is involved in the regulation of store-operated calcium influx in salivary gland cells. In the past year our major effort has continued to be towards characterizing SOCE and identifying the role of TRPC channels in agonist-stimulated calcium entry in salivary gland cells. Consistent with our previous studies, we have now reported that caveolin has a critical role in SOCE by regulating the plasma membrane assembly of TRPC1 channels. Mutation in the caveolin-binding domain of TRPC1 disrupted its plasma membrane localization and exerted a dominant negative effect on SOCE. Further, we have reported that TRPC3 interacts with SNARE proteins and that this interaction is involved in membrane trafficking of TRPC3. We have shown that TRPC3 undergoes constitutive and regulated trafficking mechanisms. Importantly, agonist-stimulated PIP2 hydrolysis, increases exocytotic insertion of TRPC3 into the plasma membrane and this contributes towards the increase in calcium entry seen in stimulated agonist-cells. Our previous studies have been largely carried out with HSG cells in which we have demonstrated conclusively that TRPC1 is the primary SOCE component. Towards characterizing SOCE components in other salivary gland cells, we have now studied SOCE in HSY cells. Our results demonstrate that distinct store-operated Ca2+ channels are present in different cells. In HSG cells, SOCE is associated with a relatively Ca2+ selective current whereas HSY cells display a non-selective cation channel. Both of these channels are distinct from the CRAC channel in RBL cells, the components of which are presently unknown. Although the physiological relevance of these different SOCE channels is not presently clear, we have now examined the molecular components of the channel in HSY cells. Interestingly, while the HSG channel appears to primarily depend on TRPC1, the HSY channel appears to be formed by the coassembly of TRPC1 and TRPC3. Further, TRPC1-TRPC3 interactions are mediated via their N-terminal domains. Expression of the NTRPC1 disrupts SOCE in HSY cells. Thus, we propose that TRCs can assemble as homomers or heteromers to form SOC and that the channel properties are defined by the specific TRPC components that are involved. To assess the physiological relevance of TRPC channels we have examined their routing in polarized epithelial cells. We have established stable TRPC expression in MDCK and salivary epithelial (SMIE) cells, both of which form high resistance monolayers when cultured on Transwell filters. We have obsevred that TRPCs have distinct cellular localization. TRPC3 is apically localized, TRPC1, TRPC5, and TRPC2 are basaly localized while TRPC6 is found in both apical and basal regions of the cell. Further, endogenous TRPC3, TRPC1, and TRPC6 were found at the same locale. We have also studied the regulation of the TRP channels in these cellular regions. Consistent with previous reports, Ca2+ signaling proteins were also predominantly localized in the apical region of these cells. Further, TRPC3 was assembled in a complex with TRPC6, but not TRPC1, and key Ca2+ signaling proteins like IP3R, G-proteins, and PLC. Importantly, we showed that TRPC3/TRPC6 channels can mediate apical calcium uptake and transepithelial calcium transport in polarized epithelial cells. These data demonstrate a novel role for TRPC3/TRPC6 channels; i.e. agonist-stimulated apical calcium uptake. Consistent with this, we detected localization of TRPC3 and TRPC6 in the apical regions of salivary gland and kidney ducts. Studies are ongoing to determine how these apically localized channels are regulated and what is their physiological function. In the coming fiscal year we will continue our studies along these directions. A major focus will directed towards determining novel TRPC interacting proteins to help us to understand their function and regulation. We will also continue to study the trafficking of TRPC channels and the mechanisms involved in thier assembly and multimerization. Stduies will also be directed identifying specific SOC currents generated by different TRPC combinations.